A The Prior Art
The large number of cloned gene products and the widespread use of DNA sequencing to determine the primary structure of proteins, has increased the need for carboxy-terminal (C-terminal) sequencing to better define post synthetic processing of proteins. C-terminal sequencing complements existing N-terminal degradations based on Edman chemistry (Edman, P. Acta. Chem. Scand. 4:283-293 (1950)). Although many methods have been proposed (see Rangarajan, M., Chemical methods of amino acid sequence analysis from carboxy terminal end. In Protein/Peptide sequence Analysis: Current Methodologies (Brown, A.S., Ed.) pp. 135-144, CRC Press, Bora Raton, Florida (1988) and Ward, C.W., Carboxyl terminal sequence analysis. In Practical Protein Chemistry----A Handbook (Darbre, A., Ed.) pp. 491-525, John Wiley and Sons, Ltd. (1986)) the thiocyanate method, first described by Schlack and Kumpf, Z. Physiol. Chem. 154:125-170 (1926), has been the most widely studied and the most attractive because of its similarity to the Edman degradation. This method involves reaction of a protein or peptide with isothiocyanate reagents, in the presence of acetic anhydride, to form a C-terminal thiohydantoin amino acid. The derivatized amino acid is then hydrolyzed to yield a shortened polypeptide and a thiohydantoin amino acid. As the thiohydantoin amino acids have similar UV absorption spectra and equivalent extinction coefficients as the phenylthiohydantoin amino acids formed during the Edman degradation, the sensitivity of the thiocyanate method is expected to be similar to that of current N--terminal methods (10-200 pmol, 20-30 cycles). Historically, the main disadvantages of this procedure have been the severity of the conditions required for complete derivatization of the C-terminal amino acid and for hydrolysis of the derivatized amino acid to yield a shortened peptide and thiohydantoin derivative. Although several groups have tried to reduce the severity of the hydrolysis conditions (Waley, et al. J. Chem. Soc. 1951:2394-2397 (1951); Kjaer, et al. Acta Chem. Scand. 6:448-450 (1952); Turner, et al. Biochim. Biophys. Acta. 13:553-559 (1954), it was not until the introduction of acetohydroxamate (Stark, G.R. Biochemistry 7:1796-1807 (1968) as a mild and rapid cleavage reagent, that the hydrolysis problem appeared solved. The introduction of trimethylsilylisothiocyanate (TMS--ITC) (see U.S. Pat. No. 4,837,165) improved the yield of thiohydantoin formation and reduced the number of complicating side products. However, even with these improvements the repetitive yields were low, limiting the number of degradation cycles to 2 or 3 residues, and certain amino acids were reported to be unable to form thiohydantoins (Hawke, et al. Anal. Biochem. 166:298-307 (1987); Miller, et al., Techniques in Protein Chemistry (Hugli, T.E., Ed.) pp. 67-78, Academic Press, Inc. (1989)).
Recent work, Bailey, et al. Biochemistry 29:3145-3156 (1990), found that hydrolysis with acetohydroxamate led to the formation of a shortened peptide with a stable hydroxamate ester at the C-terminus, thereby preventing further degradation and explaining the low repetitive yields obtained with this reagent (Miller, supra; Meuth, et al., Biochem. 21:3750-3757 (1982)). Hydrolysis with dilute aqueous triethylamine was found to lead to the quantitative formation of a thiohydantoin amino acid and a shortened peptide capable of continued degradation. Additional work (Bailey, supra) addressed the generality of the thiocyanate method by examining the reaction of TMS--ITC with model peptides containing most of the naturally occurring amino acids. Problems were identified when Pro, Asp, Glu, Thr, and Asn were encountered during the degradation. Minimization of the reaction time with acetic anhydride was found to allow the quantitative degradation of Glu and Thr and addition of a nucleophile prior to derivatization by TMS--ITC was used to allow partial degradation of C-terminal Asp.
Automation of C-terminal chemistry has been attempted by several groups Application of this chemistry to the solid phase (polypeptides covalently attached to a solid support) has been recognized to facilitate the successful automation of this chemistry. The advantages of covalently immobilizing polypeptide to a solid support include: elimination of sample washout thereby resulting in high initial and repetitive yields, the ability to use reagents and solvents optimal for derivatization and washing, and the ability to efficiently wash the sample to remove reaction by--products resulting from thiohydantoin formation, thereby creating a potential for a low chemical background.
The concept of solid phase sequencing for N--terminal Edman chemistry was first proposed by Laursen, R.A. Eur. J. Biochem 20:89-102 (1971), and has since been used successfully by a number of groups for the Edman degradation (Laursen, et al. FEBS Lett. 21:67-70 (1972); L,Italien, et al. Anal. Biochem. 127:198-212 (1982); L,Italien, Methods in Protein Microcharacterization, (Shively, J.E., Ed.) pp. 279-314, Humana press, Inc. (1986)). Initial attempts at C-terminal sequencing using the thiocyanate chemistry from covalently attached peptides was made by several groups. Williams, et al. FEBS Lett. 54:353-357 (1975) were able to perform 1-3 cycles on peptides (1 .mu.) covalently attached to N--hydroxysuccinimide activated glass beads using 12 N HCl for cleavage of the peptidylthiohydantoins. Utilizing this same procedure, Rangarajan, et al. Biochem. J. 157:307-316 (1976) were able to perform six cycles on ribonuclease (1 .mu.) covalently coupled to glass beads with a cycle time of 5 to 6 hours. Three successful cycles, with HPLC identification of the released amino acid thiohydantoins, were performed by Meuth et al. Biochem. 21:3750-3757 (1982) on a 22--amino acid polypeptide (350 nmol) covalently linked to carbonyldiimidazole activated aminopropyl glass. These authors used thiocyanic acid for derivatization to a peptidylthiohydantoin and acetohydroxamate for cleavage further reducing the time per cycle to 3 hours. A more recent report by Inglis et al., Methods in Protein Sequence Analysis (Wittmann--Lebold, B., Ed.) pp. 137-144, Springer--Verlag (1989) reports the sequential degradation of nine residues from a synthetic decapeptide (30 nmol) covalently coupled to glass beads with a cycle time of 48 min., however no experimental details were given.
Applicants initial work concerning the automation of the thiocyanate chemistry for the purpose of carboxy--terminal sequencing of proteins and peptides revealed several problems: (1) cleavage of the peptidylthiohydantoin with acetohydroxamate resulted in a shortened peptide blocked to further sequence degradation from the C-terminus due to the formation of a stable hydroxamate ester, (2) although cleavage of the peptidylthiohydantoin with dilute aqueous triethylamine does not result in the formation of a blocked shortened peptide, the use of aqueous solutions on peptides covalently bound to polyvinyldifluoride (PVDF) was found to inadequately "wet" the support and cleavage did not occur (50.degree. C.) or only very slowly at elevated temperatures (70.degree. C.). The inclusion of 30% water miscible organic solvent to the dilute triethylamine solution was found to allow adequate "wetting" of the membrane support, but significantly inhibited the cleavage reaction (Bailey, et al., Carboxy terminal sequencing: Automation and application to the solid phase, Techniques in Protein Chemistry: II (Villafranca, J.J., Ed.) pp. 115-129, Academic Press, Inc. (1991), (3) the activation of the C-terminal carboxylic acid with acetic anhydride was found to prevent or contribute to the reduced yields of sequencing on certain of the twenty naturally occurring amino acids (Bailey, et al., supra (1990), e.g., threonine, serine, aspartate, glutamate, and proline.
The cleavage problems were solved with the introduction of a new reagent, sodium trimethylsilanolate, for cleavage of the covalently coupled peptidylthiohydantoins and the introduction of a carboxylic acid modified polyethylene support for covalent attachment of the peptide sample (see, e.g., patent application PCT/US90/02723). This novel cleavage reagent was found to hydrolyze specifically the peptidylthiohydantoin into a shortened peptide capable of continued sequencing from the C-terminus and a thiohydantoin amino acid. The thiohydantoin amino acids are then identified by HPLC.